IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/20

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dspB Track

Plate result: 9 colonies

Colony PCR

PCR Master mix
Reagent1x rxn volume (uL)
5x rxn buffer5
10mM dNTP0.5
sdH2O12.15
Phusion polymerase0.1
MgCl22
DMSO - 5%1.25
10uM fw primer (VF2)2
10uM re primer (VR)2
Total25

PCR Cycles:

  • 98C @ 3min
  • Cycle 27x:
    • 98C @ 10 sec
    • 72C @ 30 sec
    • 72C @ 40 sec
    • 72C @ 10 min
  • 10C @ hold

Start: 12:54pm
End: 2:02pm

Phusion: VF2 - 64C; VR - 64C

Gel verification colony PCR products

  • Protocol: gel verification protocol in Protocol (SOP)
  • Machine conditions: 0.5x TBE buffer, 100V, 45min

Gel orientation:

Gel orientation
ABCDE100bp ladderFGHIW1 kb ladder

Results:
[[Image:‎|300px]]

O/N culture:

  • Protocol: SOP
    • With 5mL of culture with chlor 1 (A,B,C,D,G,H) in 37C at 4:54pm

Transformation (for His) again

  • 9pm
  • no colonies from yesterday
  • changes:
  1. Use 7uL of ligation mix
  2. Use 400uL of SOC instead of LB Broth

PCR using Rafael's method Attempt #4

PCR Master mix
Reagent1x rxn volume (uL)
buffer
10mM dNTP1
sdH2O31.5
Polymerase
MgCl22
DMSO - 5%2.5
gDNA1
fw rxn 23
re rxn 23
Total45
  • Gave to Rafael to finish.


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